HSC Exam Practice

Sample response. A colony forming unit (CFU) is one viable cell (or cluster of cells that were not fully separated during dilution or spreading) from the original sample that grows into a single visible colony on an agar plate. Colony counts are estimates rather than exact counts because: (1) a single colony may have grown from a cluster of two or more bacteria that were not separated rather than from one individual cell, meaning the count underestimates total cell number; and (2) the plate count only detects bacteria capable of growing on that specific medium under the specific temperature and time conditions used — dead cells, cells requiring different media, and non-bacterial microorganisms are invisible to the method.

Marking notes. 1 mark: correct definition of CFU as a viable cell or cluster that grows into one colony. 1 mark: explains that one colony may originate from more than one cell (clusters not fully separated). 1 mark: explains that the method only detects organisms that can grow under the test conditions (not dead cells, not organisms requiring different conditions).

Sample response. The negative control (sterile distilled water plated on agar) tests for contamination of the growth medium or equipment: it should show zero colonies; any growth means external contamination has occurred and invalidates the experiment. The positive control (a known bacterial suspension plated on agar) tests whether the growth medium and incubation conditions support bacterial colony formation: it should show predictable colony growth; if it shows no growth, the medium may be defective or the conditions are incorrect. The two controls test opposite failure modes: the negative tests for false positives from contamination; the positive tests for false negatives from a failed medium.

Marking notes. 1 mark: correct description of negative control function (detects contamination of medium/equipment; should give 0 colonies). 1 mark: correct description of positive control function (confirms medium supports growth; should give expected colony growth). 1 mark: explicitly distinguishes the two purposes (negative detects contamination; positive detects failed medium/conditions — opposite failure modes).

Sample response. E. coli is used as an indicator organism because its presence in water reliably signals faecal contamination, meaning that other faecal pathogens — including Salmonella, Campylobacter, and Cryptosporidium — are likely also present. Testing for every possible pathogen would be impractical, time-consuming, and expensive. E. coli is easy to culture on selective media (such as MacConkey agar), straightforward to identify and count, and provides a reliable proxy for the entire spectrum of faecal contamination risk in a single, cost-effective test.

Marking notes. 1 mark: E. coli is a reliable indicator of faecal contamination, implying the presence of other faecal pathogens. 1 mark: testing individually for every pathogen is impractical/expensive/time-consuming. 1 mark: E. coli is practical to culture on selective media and straightforward to count, making it a cost-effective proxy. (Accept any three of these points for 3 marks.)

Sample response. Serial dilution reduces the concentration of bacteria in a sample stepwise (typically 1:10 per step) so that when a measured volume is spread onto an agar plate, the resulting number of colonies falls within a range where individual colonies can be counted. Without dilution, heavily contaminated samples produce confluent (lawn) growth in which colonies overlap and cannot be distinguished. Only plates with 30–300 colonies are used because: below 30, random statistical variation has a proportionally large effect on the estimate (e.g. missing one colony changes the result by over 3%); above 300, colonies are too crowded to distinguish individually and multiple CFUs may coalesce into a single apparent colony, causing the count to underestimate true concentration.

Marking notes. 1 mark: serial dilution reduces concentration to produce countable colonies and prevents confluent growth. 1 mark: below 30 — random variation is proportionally too large / statistically unreliable. 1 mark: above 300 — colonies too crowded to distinguish / risk of merged colonies causing undercount.

Sample response. Validity refers to whether an investigation measures what it claims to measure — a valid microbial investigation uses appropriate controls, the correct selective medium for the target organism, and aseptic technique to ensure observed colonies came from the sample. One procedural decision that maximises validity: including a negative control plate (sterile water only) that should show zero colonies, confirming that any colonies on sample plates originated from the sample and not from contamination. Reliability refers to the consistency and repeatability of results across multiple measurements. One procedural decision that maximises reliability: preparing at least three replicate plates per dilution per sample, which allows averaging of counts and detection of outlier results caused by random variation in spreading or incubation.

Marking notes. 1 mark: correct definition of validity (measures what it claims; appropriate medium, controls, aseptic technique). 1 mark: specific validity-maximising procedure with explanation (e.g. negative control; correct selective medium; aseptic technique). 1 mark: correct definition of reliability (consistency/repeatability of results). 1 mark: specific reliability-maximising procedure with explanation (e.g. multiple replicate plates; standardised volumes; consistent incubation conditions).

Sample response. After a school microbial testing investigation, all used agar plates must be decontaminated before disposal — either autoclaved at 121°C for at least 15 minutes, or fully submerged in 10% bleach solution for at least 30 minutes. Plates must never be opened after incubation. These precautions are necessary because water samples can yield cultures of unknown microorganisms; even if students only intended to grow non-pathogenic environmental bacteria, the actual cultures growing on the plates may include opportunistic pathogens. Opening plates would release aerosols that could be inhaled or contaminate benches, while disposing of plates without decontamination risks releasing viable, potentially pathogenic cultures into the waste stream.

Marking notes. 1 mark: correct decontamination method stated (autoclave 121°C/15 min OR 10% bleach/30 min — accept either or both). 1 mark: states plates must not be opened after incubation (accept “do not open lids”). 1 mark: explains the risk that makes these precautions necessary (unknown/potentially pathogenic organisms may be cultured; aerosol/environmental contamination risk). All three marks require the risk to be linked to the precaution.

Sample response (a). The 10−3 dilution should be used. The 10−1 plates show confluent growth — a continuous lawn of bacteria in which individual colonies cannot be distinguished, so no count is possible. The 10−2 plates each show approximately 340–362 colonies, which exceed the upper limit of the 30–300 countable range; above 300 colonies, individual colonies are too crowded to count reliably and merged colonies risk producing an underestimate. Only the 10−3 plates (35–41 colonies each) fall within the 30–300 countable range.

Marking notes (a). 1 mark: correctly selects 10−3. 1 mark: explains 10−1 is confluent (uncountable). 1 mark: explains 10−2 exceeds 300 (too crowded / above countable range). (3 marks total for part a.)

Sample response (b). Mean colony count (10−3 plates): (38 + 41 + 35) ÷ 3 = 114 ÷ 3 = 38 colonies. CFU/mL = mean colonies ÷ (volume plated × dilution factor) = 38 ÷ (0.1 × 10−3) = 38 ÷ 0.0001 = 380,000 = 3.8 × 10⁵ CFU/mL.

Marking notes (b). 1 mark: correct mean (38). 1 mark: correct CFU/mL calculation with formula shown (3.8 × 10⁵ or 380,000 CFU/mL). (2 marks total for part b.)

Sample response (c). Converting: 3.8 × 10⁵ CFU/mL × 100 mL = 3.8 × 10⁷ CFU/100 mL. This vastly exceeds the guideline of fewer than 1 CFU E. coli/100 mL. This water is not safe for drinking. One further piece of information needed before a final recommendation: the result of a negative control plate — if the negative control shows any colony growth, the investigation is invalid (equipment/medium contamination cannot be excluded) and the test must be repeated with sterile materials before action is taken. (Accept also: confirmation from a second independent test / accredited laboratory; information on whether the MacConkey agar was freshly prepared and within date; whether aseptic technique was properly observed.)

Marking notes (c). 1 mark: correct conversion to per 100 mL (3.8 × 10⁷). 1 mark: states the water is unsafe with reference to the guideline. 1 mark: identifies a specific, relevant further piece of information needed (negative control result; accredited lab confirmation; or equivalent). (2 marks total for part c.)

Sample response. The serial dilution and plate count method is an invaluable tool for assessing drinking-water safety in remote Australian communities, but it has well-defined strengths and limitations that must be understood when interpreting results.

The method’s primary strength is that it provides a quantitative estimate of viable bacterial concentration in CFU per mL or per 100 mL, allowing direct comparison against the Australian Drinking Water Guideline threshold of fewer than 1 CFU of E. coli per 100 mL. Using E. coli as the indicator organism is particularly effective: its presence reliably signals faecal contamination, implying that other faecal pathogens including Salmonella, Campylobacter, and Cryptosporidium may also be present. The method is relatively simple, inexpensive, and can be performed in field or regional laboratory settings appropriate for remote Australia. Using serial dilution and counting only plates with 30–300 colonies provides a reliable estimate of bacterial load across a wide range of initial concentrations.

However, the method has significant limitations in detecting all health hazards. Nutrient or selective agar plate counts detect only bacteria capable of growing on that specific medium under the incubation conditions used. Viruses — including enteric viruses such as norovirus and hepatitis A virus — cannot be cultured on agar and would yield zero colonies regardless of concentration. Protozoan parasites such as Cryptosporidium parvum and Giardia lamblia are also undetectable by this method; they require immunofluorescence microscopy or PCR-based assays. A zero E. coli count therefore does not guarantee freedom from viral or protozoal contamination — an important distinction in remote communities where children under five suffer disproportionately from waterborne illness. Additionally, a single quarterly sample may miss contamination events caused by rainfall, flooding, or pipe disturbance occurring between collection dates.

When results exceed the guideline — as documented in up to 40% of remote community water supplies in Australia (ANU, 2021) — the public health significance is serious. A count above 1 CFU E. coli/100 mL indicates confirmed faecal contamination; the contamination source may be a damaged bore casing, livestock access to the water supply, or inadequate treatment. Immediate public health responses include issuing a boil-water advisory (boiling kills bacteria and most protozoa), investigating and eliminating the contamination source, and treating the supply with chlorination or UV disinfection. Ongoing monitoring is essential to confirm that intervention is effective.

In summary, the serial dilution and plate count method is a sound, practical, and internationally validated tool for bacterial safety screening. Its value is real but scoped: it detects faecal bacterial contamination reliably, but must be complemented by other methods (viral assays, protozoan detection) for a complete public health risk assessment.

Marking notes. 1 mark — States at least two specific strengths of the method (quantitative CFU result enabling comparison to guideline; E. coli as reliable faecal indicator; practical/affordable). 1 mark — Identifies viruses as a class of pathogen not detected by the plate count method, with a named example and a correct reason (cannot grow on agar; require living host cells). 1 mark — Identifies protozoa as a class of pathogen not detected, with a named example and a correct reason (require different detection method). 1 mark — Explains that a zero plate count does not confirm the water is free of all pathogens (links to the plate count limitation). 1 mark — States the public health significance of a result above the Australian Drinking Water Guideline (confirmed faecal contamination; implied presence of other pathogens; specific consequence for health in remote communities). 1 mark — Describes at least one specific, justified immediate public health action linked to lesson content (boil-water advisory; source investigation and treatment; chlorination). 1 mark — Reaches an explicit evaluative judgement that is nuanced and not a simple “the method is good/bad” — judges it as a valuable but limited screening tool that must be complemented by additional methods for a complete risk assessment.